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PubMed1999

Evaluation of the pharmacokinetic features and tissue distribution of the potent nonnucleoside inhibitor of HIV-1 reverse transcriptase, N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240) with an analytical HPLC method.

PURPOSE: The purpose of the present study was to examine the pharmacokinetic features and tissue distribution of N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240), a novel non-nucleoside inhibitor of HIV reverse transcriptase with potent anti-viral activity against AZT-sensitive as well as multidrug-resistant HIV-1 strains. METHODS: A sensitive and accurate high performance liquid chromatography (HPLC)-based quantitative detection method was established to measure concentrations of HI-240 in pharmacokinetic studies. The plasma concentration-time data were modeled by using the WinNonlin program to estimate the pharmacokinetic parameter values. RESULTS: HI-240 had an elimination half-life of 78.3 +/- 2.0 min after i.v. administration and 196.8 +/- 3.1 min after i.p. administration. The systemic clearance of HI-240 was 2194 +/- 61 ml/h/kg after i.v. administration and 9339 +/- 1160 ml/h/kg after i.p. administration. Following i.v. injection, HI-240 rapidly distributed to and accumulated in multiple tissues with particularly high accumulation in adipose tissue, adrenal gland, and uterus+ovary. The concentration of HI-240 in brain tissue was comparable to that in the plasma, indicating that HI-240 easily crosses the blood-brain-barrier. Following i.p. injection, HI-240 was rapidly absorbed with a t1/2ka and a tmax values of less than 10 min. Following oral administration, HI-240 was absorbed with a t1/2ka of 4.2 +/- 1.1 min and a tmax of 95.1 +/- 25.1 min. The intraperitoneal bioavailability was estimated at 23.5%, while the oral bioavailability was only 1%. CONCLUSIONS: The HPLC-based accurate and precise analytical detection method and pilot pharmacokinetic studies described herein provide the basis for advanced preclinical pharmacodynamic studies of HI-240. The ability of HI-240 to distribute rapidly and extensively into extravascular compartments and easily cross the blood-brain barrier represent significant pharmacokinetic advantages over AZT.

Administration, Oral↗
PubMed1995

Truncating alpha-helix E' of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer.

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.

Amino Acid Sequence↗
PubMed1993

Identification of the amino acid in the human immunodeficiency virus type 1 reverse transcriptase involved in the pyrophosphate binding of antiviral nucleoside triphosphate analogs and phosphonoformate. Implications for multiple drug resistance.

A recombinant clone of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with reduced sensitivity to 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) and phosphonoformate (PFA), a pyrophosphate analog, has been obtained from the RNA of HTLV-IIIB infected cells using the polymerase chain reaction. The mutant HIV-1 RT retained polymerase activity and was cross-resistant to triphosphate forms of other nucleoside analogs including 2',3'-dideoxycytidine 5'-triphosphate, 2',3'-dideoxyadenosine 5'-triphosphate, and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (D4TTP), but remained sensitive to the non-nucleoside HIV-1 RT inhibitors, such as nevirapine and TIBO R82150. Sequence analysis of the mutant HIV-1 RT revealed a single amino acid substitution (Val-->Ala) at amino acid 90. The substitution of amino acid 90 by the closely related amino acids, such as Thr and Gly, also showed decreased sensitivity to AZTTP, D4TTP, and PFA. All these mutations at amino acid 90 also caused an alteration of Km for thymidine triphosphate. These results suggest that Val at this site plays a role in determining the interaction of the HIV-1 RT enzyme with the pyrophosphate group of deoxynucleoside triphosphate (dNTP) and that the hydrophobicity of the amino acid at this position was the most important determinant in the binding of HIV-1 RT to dNTP.

Amino Acids↗
PubMed1999

Nitric oxide inhibits the HIV-1 reverse transcriptase activity.

Nitric oxide (NO) is a polypotent regulatory molecule involved in a variety of activities, such as the modulation of the catalytic activity of cysteine-containing enzymes. The present study reports the modulation of the HIV-1 reverse transcriptase activity by NO, released by the NO-donors 3, 3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), 4-(phenylsulfonyl)-3-((2-(dimethylamino) ethyl)thio)furoxan oxalate (SNO-102), and sodium nitroprusside (SNP). NO inhibits dose-dependently the HIV-1 reverse transcriptase activity, likely due to oxidation of Cys residue(s). Present results, representing a new insight into the modulation mechanism of the HIV-1 reverse transcriptase activity, may be relevant to develop new strategies for inhibition of HIV-1 replication.

Catalysis↗
PubMed2002

Altering the RNase H primer grip of human immunodeficiency virus reverse transcriptase modifies cleavage specificity.

Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic center. Within the RNase H domain, these residues include Thr473, Glu475, Lys476, Tyr501, and Ile505, while His539 and Asn474 interact with the scissile phosphate of the RNA template. Amino acid substitutions at several of these positions were evaluated in the context of hydrolysis of nonspecific RNA-DNA hybrids and substrates mimicking specific RNase H-mediated events. With the exception of mutant I505G, which exhibited a dimerization defect, substituting alanine at positions 473-476 and 501 had minimal consequences for DNA synthesis on duplex and hybrid DNA and RNA substrates. In contrast, the efficiency with which most mutants catalyzed polymerization-independent RNase H cleavage was sharply reduced. This deficiency was more pronounced when mutant enzymes were challenged to process the (+) strand polypurine tract (PPT) primer from either (+) RNA or a PPT/(+) DNA RNA/DNA chimera. Reduced polymerization-independent RNase H activity also significantly influenced the rate of DNA strand transfer, suggesting the donor template must be reduced in size below 13 nt before this event proceeds.

Amino Acid Sequence↗
PubMed1996

Reverse transcriptase mutations in HIV-1-infected children treated with zidovudine.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutations were detected in DNA from peripheral blood mononuclear cells from 11 of 12 HIV-1-infected children after 11-20 months of zidovudine monotherapy. The number of children with mutations detected at each codon were as follows: codon 41, 4; codon 67, 2; codon 70, 7; codon 215, 7; codon 219, 0. Codon 41 mutations were found only in the presence of a codon 215 mutation and in the absence of a codon 70 mutation. The codon 41/215 mutant combination was associated with decline in weight-for-age z score during therapy, weight < 10th percentile, CD4+ cell counts < 3rd percentile, and immune-complex dissociated HIV-1 p24 antigen (ICD p24 Ag) levels > 100 pg/ml. Patients developing the codon 70 mutation tended to have body weight > 30th percentile, CD4+ cell counts > 25th percentile, and ICD p24 Ag < 100 pg/ml. The codon 41 mutation was associated with clinical deterioration during a 6-month followup period.

Anti-HIV Agents↗
PubMed1994

Deoxynucleotide polymerization by HIV-1 reverse transcriptase is terminated by site-specific styrene oxide adducts after translesion synthesis.

In an effort to integrate an understanding of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) structure with its function, the action of HIV-1 RT was examined in vitro on DNA templates modified with a model bulky DNA adduct. Styrene oxide was site-specifically and stereospecifically coupled to the N6 position of adenine to form six different adducted templates. Primer extension assays were conducted under conditions defining both single and multiple encounters between the polymerase and the damaged template-primer. The extent of polymerization observed for each adduct was found to depend on both the chirality of the damage and the lesion sequence context. When HIV-1 RT polymerization was limited by single encounters with damaged DNA, the SO lesions were readily bypassed as evidenced by minimal pausing at the adducted base. However, RT replication of all SO-modified templates was significantly terminated 3-5 nucleotides after translesion synthesis but before reaching the end of the template. These truncated products could be readily extended when additional encounters between enzyme and template-primer were permitted. A model is presented to explain these results in the context of HIV-1 RT structure during DNA replication.

Base Sequence↗
PubMed1996

The structure of an RNA/DNA hybrid: a substrate of the ribonuclease activity of HIV-1 reverse transcriptase.

The structure of a complementary hybrid duplex of RNA and DNA has been determined by X-ray crystallography. A ten residue DNA oligonucleotide of sequence 5'-G-G-C-G-C-C-C-G-A-A-3' was annealed to complementary RNA (5'-u-u-c-g-g-g-c-g-c-c-3') and crystallized, producing tetragonal crystals that diffract to 2.3 A resolution. The hybrid adopts a geometry that is neither strictly A nor B-form, rather the helix possesses qualities of both, reminiscent of spectroscopic descriptions of a hybrid conformation, or H-form. All of the ribonucleotides maintain the C3'-endo conformation seen in A-form, while both C3'-endo and C2'-endo conformations are found in the deoxyribonucleotides. The minor groove width (8.5 to 10.5 A) is intermediate between standard values for A (11 A) and B-form (7.4 A) DNA. The global parameters rise and base-pairs tilt (or inclination) are like that of A-DNA, however the slide and x displacement (Dx) are more like that of A-RNA, thus giving the hybrid a unique conformation. In addition, the 10-mer crystallizes in a manner that allows the formation of dimers that stack end-to-end, thereby providing a glimpse of how an extended (20 base-pair) helix of RNA-DNA hybrid might appear. This duplex sequence was selected for study because it is specifically recognized by the ribonuclease H function of HIV reverse transcriptase. A structure of a substrate of this enzyme is of potential value in understanding requirements for the selectivity of this important drug target. The minor groove of the hybrid duplex, lined with the 2-OH of the ribose rings, is the single distinguishing characteristic of the RNA/DNA hybrid, undoubtedly an important structural feature conferring selectivity.

Crystallography, X-Ray↗
PubMed2006

A sensitive genotyping assay for detection of drug resistance mutations in reverse transcriptase of HIV-1 subtypes B and C in samples stored as dried blood spots or frozen RNA extracts.

Exposure to antiretroviral drugs in resource-constrained settings is likely to result in the emergence of drug-resistant HIV-1 variants, limiting treatment options. Genotypic drug resistance testing assists clinical management and outcomes assessment, but a sensitive and reproducible genotypic assay feasible for resource-constrained settings is needed. A sensitive, reproducible genotyping assay to detect HIV-1 drug resistance mutations in reverse transcriptase and protease was developed and validated using blood stored as dried blood spots or as frozen RNA extracts from Zambian children infected with subtype C. HIV-1 genotypes derived from samples stored as dried blood spots were compared to those derived from paired liquid plasma samples in American young adults infected with HIV-1 subtype B. The method reproducibly amplified patient-specific sequences and detected drug resistance mutations from all of the dried blood spots or excess frozen RNA extracts with detectable viremia over a broad range of viral loads (193-3 million HIV-1 RNA copies/mL) in both HIV-1 subtypes B and C infection. This method captured the genetic variation typical of HIV-1 infection, including mutations at usual sites of drug resistance, polymorphisms, and mixtures. This sensitive and reproducible genotypic assay is feasible for detection of antiretroviral resistance in resource-constrained settings.

Adolescent↗
PubMed2002

Nontemplated nucleotide addition by HIV-1 reverse transcriptase.

We studied the kinetics of nontemplated nucleotide addition by the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) using model substrates derived from the 3' end of HIV-1 minus-strand strong-stop DNA. The addition of a nontemplated nucleotide was highly dependent on the nature of the base (fastest addition with dATP), type of nucleoside, and pH of the reaction buffer. The salt concentration, presence or absence of nucleocapsid protein, and nature of the blunt-ended duplex (DNA/DNA versus RNA/DNA) had only limited effects. The efficiency and base specificity were strongly affected by the sequence at the 3' end of the blunt-ended duplex. In every case, nontemplated nucleotide addition was much slower than templated polymerization. The K(d) for the incoming dNTP with an RT bound to a blunt-ended duplex was at least 1000-fold higher than with a duplex with a template overhang. At concentrations normally found in vivo, ATP can compete with dNTPs for binding to the polymerase active site and reduce the efficiency of nontemplated nucleotide addition. Although a stable ternary complex RT/DNA/dNTP could be readily detected by gel retardation assays if the DNA had a template overhang, stable ternary complexes were not observed with a blunt-ended duplex substrate. At in vivo concentrations of dNTPs (5-10 microM), nontemplated nucleotide addition occurred, but it was very inefficient and the rate of nontemplated polymerization is at least 10000-fold slower than the rate of templated polymerization. We could conclude that, in vivo, the unfavorable binding of the incoming dNTP, low concentration of dNTPs, the presence of a large concentration of ATP, and the inability to form a stable ternary complex prior to the polymerization step collaborate to reduce the efficiency of nontemplated nucleotide addition.

Adenosine Triphosphate↗
PubMed1991

Localization of a polynucleotide binding region in the HIV-1 reverse transcriptase: implications for primer binding.

Properties of primer recognition by purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) p66 homodimer have been investigated. Earlier studies had shown that RNA-directed DNA synthesis catalyzed by HIV-1 RT proceeds by an ordered mechanism in which template-primer combines with the free enzyme to form the first complex in the reaction scheme, and it was also shown that primer alone is a competitive inhibitor of template-primer. In this study, enzyme-primer binding has been further characterized utilizing pd(T)8 and pd(T)16 as model primers and UV cross-linking to covalently trap the enzyme-primer complexes. Competition experiments with several authentic primers, including tRNA(3Lys), indicate that pd(T)n binds to the kinetically significant primer binding site of RT. Salt reversal experiments suggested that the free energy of pd(T)n binding to RT has a large nonelectrostatic component. Binding of pd(T)n to p66-RT is not affected by dNTPs and does not require the presence of template. The site of UV cross-linking of pd(T)16 was localized to the NH2-terminal half of p66 by use of V8 protease hydrolysis and microsequencing. Our results indicate that a polynucleotide binding site is in close proximity to residues in the peptide comprising amino acids 195 approximately 300. This region could be either a single-stranded template or single-stranded primer binding site; however, we have documented the specificity of binding with oligonucleotides that act as primer in the in vitro DNA synthesis reaction. Therefore, this d(T)16 binding site may be part of a primer-binding groove within the HIV-1 reverse transcriptase.

Animals↗
PubMed1999

Rapid detection of the HIV type 1 reverse transcriptase codon T215Y by reverse transcription-polymerase chain reaction with fluorogenic probes.

Early detection of viral mutations, particularly those mutations associated with cross-resistance to antiretroviral drugs, is critical both for understanding the mechanism of drug resistance and for the clinical management of patients infected with HIV-1. One of the frequently observed mutations in the HIV-1 reverse transcriptase (RT)-coding region is ACC --> TAC at codon 215, resulting in a change of wild-type threonine (T) to tyrosine (Y); this mutation has been associated with decreased phenotypic susceptibility to zidovudine (ZDV). We describe a technique for the detection of the T215Y mutation using reverse transcription-polymerase chain reaction (RT-PCR) amplification of viral sequences and a 5' nuclease assay requiring fluorogenic probes. In addition to detecting the presence of the ACC --> TAC mutation at codon 215, this assay provides an increased ability to detect low levels of mutant species in a mixed population, relative to conventional sequencing. Further advantages of this technique include the rapid and high-throughput nature of the assay, the accuracy of the assay relative to conventional DNA sequencing, and the convenience of combining RT-PCR virus amplification with the allelic discrimination assay, without the need for purification of PCR products.

Anti-HIV Agents↗
PubMed2005

Polymorphism and drug-selected mutations in the reverse transcriptase gene of HIV-2 from patients living in southeastern France.

Few data are available about the susceptibility and the genotypic resistance pattern of human immunodeficiency virus type 2 (HIV-2) to nucleoside reverse transcriptase inhibitors (NRTIs). The HIV-2 reverse transcriptase (RT) gene from 25 HIV-2-infected patients followed-up in Marseilles and the surrounding area was analyzed. The aims of this study were to characterize the polymorphism of HIV-2 RT in the absence of drug, to determine whether it naturally harbors codons associated with drug-resistance in HIV-1, and to identify mutations emerging under NRTI-selective pressure. Fourteen patients had never undergone antiretroviral therapy and 11 received NRTI. Seventy sequences were analyzed. In untreated patients, 12 spots of high natural polymorphism (at positions 10, 11, 20, 43, 104, 121, 135, 162, 176, 180, 200, and 227) were observed; 4 of them were specific of HIV-2 (10, 176, 180, 227). Moreover, results showed four positions that could be associated with natural resistance to NRTI (75I, 118I, 219E, and perhaps 215S), in addition to those described previously for non-nucleoside reverse transcriptase inhibitors (NNRTIs) (181I, 188L, 190A). In HIV-2-infected patients receiving NRTI-containing therapies, specific genotypic patterns were observed with a high frequency of mutation Q151M (in 45% of patients) often associated with 70R, 115F, 214L, and/or 223R, which might compose an HIV-2 multi-NRTI resistance complex. Four newly or rarely described NRTI-selected mutations were observed: I5V, K35R, F214L, and K223R. As in HIV-1, substitution M184V was found in 3TC-treated patients. In conclusion, these findings highlight the need for specific guidelines for determining genotypic resistance and treatment of HIV-2.

Adult↗
PubMed2000

Role of a dipeptide insertion between codons 69 and 70 of HIV-1 reverse transcriptase in the mechanism of AZT resistance.

The 3'-azido-3'-deoxythymidine (AZT)-resistant pheno type of a heavily mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) carrying a dipeptide (Ser-Ser) insertion between codons 69 and 70 as well as other mutations related to resistance to RT inhibitors has been studied. Recombinant virus carrying this variant RT (termed SS RT) showed reduced susceptibility to all nucleoside RT inhibitors in clinical use, particularly to AZT. In the presence of ATP, recombinant SS RT had an increased ability to remove the 3'-terminal nucleotide from AZT- terminated primers and extend the unblocked primer, compared with wild-type HIV-1 RT (BH10 isolate). Insertion of two serines in the sequence context of BH10 RT did not affect the ATP-dependent phosphorolytic activity of the enzyme, and had no influence in resistance to RT inhibitors. However, SS RT mutants lacking the dipeptide insertion or bearing a four-serine insertion showed reduced ATP-dependent phosphorolytic activity that correlated with increased AZT sensitivity, as determined using a recombinant virus assay. Therefore, the insertion appears to be critical to enhance AZT resistance in the sequence context of multidrug-resistant HIV-1 RT.

Amino Acid Sequence↗
PubMed1992

Template. Phosphorothioate oligonucleotides duplexes as inhibitors of HIV-1 reverse transcriptase.

We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).(dT)14 as template primer. In the second, the oligonucleotides or their duplexes were used to displace a fluorescent template primer complex of known affinity from its binding site on reverse transcriptase. The two methods gave the same relative order of affinity. Phosphorothioate oligodeoxyribonucleotides had a much higher affinity than oligo(dC)14 and it was increased on hybridization. Quantitatively similar results were obtained for S(dC)14 or its analog with bases in the alpha-configuration. Of the analogs tested, only S(dC)14 showed priming activity.

Antiviral Agents↗